Methods for sterilizing preparations of glycosidases

Abstract

Methods are disclosed for sterilizing preparations of glycosidases to reduce the level therein of one or more active biological contaminants or pathogens, such as viruses, bacteria (including inter- and intracellular bacteria, such as mycoplasmas, ureaplasmas, nanobacteria, chlamydia, rickettsias), yeasts, molds, fungi, single or multicellular parasites, prions or similar agents responsible, alone or in combination, for TSEs. These methods involve sterilizing preparations of glycosidases, such as alpha-glucosidase or alpha-galactosidase, with irradiation.

What is claimed is:  
     
       1. A method for sterilizing a preparation of one or more glycosidases that is sensitive to radiation, said method comprising reducing the temperature of said preparation of one or more glycosidases to a temperature below ambient temperature, and irradiating said preparation of one or more glycosidases with radiation for a time effective to sterilize said preparation of one or more glycosidases at a rate effective to sterilize said preparation of one or more glycosidases and to protect said preparation of one or more glycosidases from said radiation. 
     
     
       2. The method according to  claim 1 , said method further comprising adding to said preparation of one or more glycosidases at least one stabilizer in an amount effective to protect said preparation of one or more glycosidases from said radiation prior to said irradiating. 
     
     
       3. The method according to  claim 1 , said method further comprising reducing the residual solvent content of said preparation of one or more glycosidases to a level effective to protect said preparation of one or more glycosidases from said radiation prior to said irradiating. 
     
     
       4. The method according to  claim 1 , said method further comprising applying to said preparation of one or more glycosidases prior to irradiating at least one stabilizing process selected from the group consisting of: 
       (a) reducing the residual solvent content of said preparation of one or more glycosidases, and  
       adding an effective amount of at least one stabilizer to said preparation of one or more glycosidases.  
     
     
       5. The method according to  claim 1 , said method further comprising applying to said preparation of one or more glycosidases prior to irradiating at least one stabilizing process selected from the group consisting of: 
       (a) reducing the residual solvent content of said preparation of one or more glycosidases, and  
       adding an effective amount of at least one stabilizer to said preparation of one or more glycosidases, wherein said stabilizing processes are together effective to protect said preparation of one or more glycosidases from said radiation and further wherein said stabilizing processes may be performed in any order.  
     
     
       6. The method according to  claim 3 ,  4  or  5 , wherein said solvent is water. 
     
     
       7. The method according to  claim 6 , wherein said residual water content is reduced by the addition of an organic solvent. 
     
     
       8. The method according to  claim 3 ,  4  or  5 , wherein said solvent is an organic solvent. 
     
     
       9. The method according to  claim 3 ,  4  or  5  wherein said prepartion of one or more glycosidases is suspended in an organic solvent following reduction of said residual solvent content. 
     
     
       10. The method according to  claim 1 , wherein said effective rate is not more than 3.0 kGy/hour. 
     
     
       11. The method according to  claim 1 , wherein said effective rate is not more than 2.0 kGy/hour. 
     
     
       12. The method according to  claim 1 , wherein said effective rate is not more than 1.0 kGy/hour. 
     
     
       13. The method according to  claim 1 , wherein said effective rate is not more than 0.3 kGy/hour. 
     
     
       14. The method according to  claim 1 , wherein said effective rate is not more than 3.0 kGy/hour. 
     
     
       15. The method according to  claim 1 , wherein said effective rate is not more than 6.0 kGy/hour. 
     
     
       16. The method according to  claim 1 , wherein said effective rate is not more than 18.0 kGy/hour. 
     
     
       17. The method according to  claim 1 , wherein said effective rate is not more than 30.0 kGy/hour. 
     
     
       18. The method according to  claim 1 , wherein said effective rate is not more than 45 kGy/hour. 
     
     
       19. The method according to  claim 1 , wherein said preparation of one or more glycosidases is maintained in a low oxygen atmosphere. 
     
     
       20. The method according to  claim 1 , wherein said preparation of one or more glycosidases is maintained in an atmosphere comprising at least one noble gas. 
     
     
       21. The method according to  claim 20 , wherein said noble gas is argon. 
     
     
       22. The method according to  claim 1 , wherein said preparation of one or more glycosidases is maintained in a vacuum. 
     
     
       23. The method according to  claim 3 ,  4  or  5 , wherein said residual solvent content is reduced by a method selected from the group consisting of lyophilization, drying, concentration, addition of solute, evaporation, chemical extraction, spray-drying, vitrification and combinations of two or more thereof. 
     
     
       24. The method according to  claim 3 ,  4  or  5 , wherein said residual solvent content is less than 15%. 
     
     
       25. The method according to  claim 3 ,  4  or  5 , wherein said residual solvent content is less than 10%. 
     
     
       26. The method according to  claim 3 ,  4  or  5 , wherein said residual solvent content is less than 3%. 
     
     
       27. The method according to  claim 3 ,  4  or  5  wherein said residual solvent content is less than 2%. 
     
     
       28. The method according to  claim 3 ,  4  or  5  wherein said residual solvent content is less than 1%. 
     
     
       29. The method according to  claim 3 ,  4  or  5 , wherein said residual solvent content is less than 0.5%. 
     
     
       30. The method according to  claim 3 ,  4  or  5 , wherein said residual solvent content is less than 0.08%. 
     
     
       31. The method according to  claim 1 , wherein at least one sensitizer is added to said preparation of one or more glycosidases prior to said step of irradiating said preparation of one or more glycosidases. 
     
     
       32. The method according to  claim 1 , wherein said preparation of one or more glycosidases contains at least one biological contaminant or pathogen selected from the group consisting of viruses, bacteria, yeasts, molds, fungi, parasites, and prions or similar agents responsible, alone or in combination, for TSEs. 
     
     
       33. The method according to  claim 2 ,  4  or  5 , wherein said at least one stabilizer is an antioxidant. 
     
     
       34. The method according to  claim 2 ,  4  or  5  wherein said at least one stabilizer comprises a free radical scavenger. 
     
     
       35. The method according to  claim 2 ,  4  or  5  wherein said at least one stabilizer is a combination stabilizer. 
     
     
       36. The method according to  claim 2 ,  4  or  5 , wherein said at least one stabilizer comprises a ligand. 
     
     
       37. The method according to  claim 36 , wherein said ligand is heparin. 
     
     
       38. The method according to  claim 36 , wherein said ligand is a substrate or substrate analog of at least one glycosidase contained in said preparation of one or more glycosidases. 
     
     
       39. The method according to  claim 2 ,  4  or  5 , wherein said at least one stabilizer reduces damage due to reactive oxygen species. 
     
     
       40. The method according to  claim 2 ,  4  or  5  wherein said at least one stabilizer is selected from the group consisting of: ascorbic acid or a salt or ester thereof; glutathione; 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid; uric acid or a salt or ester thereof; methionine; histidine; N-acetyl cysteine; lipoic acid; sodium formaldehyde sulfoxylate; gallic acid or a derivative thereof; propyl gallate and mixtures of two or more thereof. 
     
     
       41. The method according to  claim 40 , wherein said mixtures of two or more additional stabilizers are selected from the group consisting of: mixtures of ascorbic acid, or a salt or ester thereof, and uric acid, or a salt or ester thereof; mixtures of ascorbic acid, or a salt or ester thereof, and 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid; mixtures of ascorbic acid, or a salt or ester thereof, uric acid, or a salt or ester thereof; and 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid; and mixtures of uric acid, or a salt or ester thereof, lipoic acid, sodium formaldehyde sulfoxylate, gallic acid or a derivative thereof, propyl gallate and 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid. 
     
     
       42. The method according to  claim 2 ,  4  or  5 , wherein said at least one stabilizer is comprises a dipeptide stabilizer. 
     
     
       43. The method according to  claim 42 , wherein said dipeptide stabilizer is selected from the group consisting of glycyl-glycine (Gly—Gly), carnosine, anserine and combinations of two or more thereof. 
     
     
       44. The method according to  claim 1 , wherein said radiation is corpuscular radiation or electromagnetic radiation, or a mixture thereof. 
     
     
       45. The method according to  claim 44 , wherein said electromagnetic radiation is selected from the group consisting of radio waves, microwaves, visible and invisible light, ultraviolet light, x-ray radiation, gamma radiation and combinations thereof. 
     
     
       46. The method according to  claim 1 , wherein said radiation is gamma radiation. 
     
     
       47. The method according to  claim 1 , wherein said radiation is E-beam radiation. 
     
     
       48. The method according to  claim 1 , wherein said radiation is visible light. 
     
     
       49. The method according to  claim 1 , wherein said radiation is ultraviolet light. 
     
     
       50. The method according to  claim 1 , wherein said radiation is x-ray radiation. 
     
     
       51. The method according to  claim 1 , wherein said radiation is polychromatic visible light. 
     
     
       52. The method according to  claim 1 , wherein said radiation is infrared. 
     
     
       53. The method according to  claim 1 , wherein said radiation is a combination of one or more wavelengths of visible and ultraviolet light. 
     
     
       54. The method according to  claim 1 , wherein said radiation is conducted at a temperature below ambient temperature. 
     
     
       55. The method according to  claim 1 , wherein said radiation is conducted below the freezing point of said preparation of one or more glycosidases. 
     
     
       56. The method according to  claim 1 , wherein said irradiation is conducted below the eutectic point of said preparation of one or more glycosidases.